CN103205406B - A kind of method utilizing subtilis 6-7 to produce heat-resisting beta-amylase - Google Patents
A kind of method utilizing subtilis 6-7 to produce heat-resisting beta-amylase Download PDFInfo
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Abstract
The invention belongs to biological technical field, a bacillus subtilis (Bacillus is provided? subtilis) 6-7 produces the diastatic method of heat-resisting B-.The bacterium being numbered 6-7 is filtered out from the soil sample and Laboratories Accession bacterial classification of nature collection.The 60h enzyme that ferments in basic medium is lived and is reached 2200.0U/mL, but in the substratum taking cassava as carbon source, fermenting enzyme work can reach 5120.5U/mL, significantly improves 2.33 times.To medium component and training systern, substratum after optimization is Tapioca Starch 2%, bean cake powder 4%, 0.1% Secondary ammonium phosphate, 0.00139% ferrous sulfate, 0.6% Trisodium Citrate, 0.4% potassium primary phosphate, 0.0005% zinc sulfate, 0.0123% magnesium sulfate, 0.0111% calcium chloride, pH7.0,37 DEG C, 190r/min, liquid amount 50ml/250ml triangular flask, initial pH7.0, inoculum size 4% shake-flask culture 60h, enzyme work can reach 6724.0U/mi.Described beta-amylase optimal reactive temperature, pH are respectively 65 DEG C, 6.0.This enzyme is widely used in the fields such as beer, food, medicine, weaving.The invention provides and utilize cassava fermentative production microorganism beta-amylase, have that cost is low, output is high, the advantage of good product performance.This bacterial strain is preserved in China typical culture collection center, does is deposit number: CCTCC? M? 209200.
Description
Technical field
The present invention relates to production bacterium and Optimization Technology method thereof that a kind of wild-type B. subtilis Bacillussubtilis6-7 produces the outer heat-resisting beta-amylase of born of the same parents, belong to enzyme engineering and fermentation engineering field.
Background technology
Beta-amylase (β-amylase, EC3.2.1.2) is also known as maltoside enzyme, glycogenase, saccharogen amylase, and its system is called α-Isosorbide-5-Nitrae-dextran-4-malto-hydrolase (α-Isosorbide-5-Nitrae-D-glucanmaltohydrolase).Beta-amylase is a kind of circumscribed-type saccharifying enzyme, when acting on starch, next maltose unit can be cut in turn from the non reducing end of α-Isosorbide-5-Nitrae glycosidic link, generate maltose and macromolecular β-limit dextrin, but can not hydrolyzing alpha-1,6-glycosidic link, runs into the tapping point of α-1,6 key, then stagnate, therefore hydrolysis rate is slower.When its hydrolyzing amylopectin, linear fraction generates maltose, and near tapping point and inner side left behind because not being hydrolyzed.During due to this zymolyte, there is Walden translocation reaction (Waldeninversion), make product become β-type maltose from α-type, therefore named beta-amylase.
β-starch is one of main saccharifying enzyme in the starch course of processing, reaction does not need coenzyme, and mild condition, by product is few, can be used as the saccharifying agent in the industrial production such as maltose, beer, beverage, have important using value in industries such as food-processing, beer fermentation, textiles, medicine, chemical industry medical analysis.Wherein microbe-derived beta-amylase, because its production cost is low, environmental hazard is little, inulinase-producing activity is high, is convenient to industrial production; Along with the development of enzyme industry and people are to the requirements at the higher level of maltose quality, application zymin produces the focus that malt syrup has become research, and the demand of beta-amylase constantly increases and makes it become one of industrial enzyme of most development prospect.
At present, β-amylase market sold has two kinds: one is microorganism (comprising bacterium and fungi) fermentative production; Another kind is that traditional technology is extracted from plant.The barley beta-amylase of plant origin is widely used in multiple field always for a long time, nearly all uses in by plant the beta-amylase obtained at present in the industry such as happy sugar, beer.Barley beta-amylase (trade(brand)name OPTLMALTBBA) as Jie Neng section is most popular, but still can not meet the demand in domestic production, is applicable to the high beta-amylase of cost performance to guarantee the need of production of high malt sugar so find.And beta-amylase is extracted from plant, technique is coarse, and enzyme activity is low, consumes a large amount of biomass energies.
Since within 1972, having delivered in microorganism and also existing and present the enzyme of mechanism of action identical with higher plant beta-amylase, had been found that many microorganisms are that beta-amylase produces bacterium, it is better than the beta-amylase of plant origin in heat-resisting.In recent years, that reports both at home and abroad is mainly contained the bacillus such as bacillus cereus (Bacilluscereus), bacillus megaterium (Bacillusmegaterium), Paenibacillus polymyxa (Bacilluspolymyxa), Bacillus circulans (Bacilluscirculans), hot anaerobic bacillus(cillus anaerobicus) genus (Thermoanaerobacterium), Rhodopseudomonas (Pseudomonassp.), streptomyces (Streptomycessp.) etc. by the bacterial strain of production by biological beta-amylase.The microorganism beta-amylase now reported is mostly middle warm type, and optimal reactive temperature is 40 DEG C ~ 60 DEG C; The beta-amylase optimal reactive temperature that hot sulphur clostridium (Clostridiumthermosulfurog) is synthesized is 75 DEG C, but it is anaerobic type bacterial strain, growth temperature is 60 DEG C of comparatively harshnesses, and be not suitable for the requirement of suitability for industrialized production, therefore the Bacillus strain of fire resistant alpha-diastase is produced in screening is then research emphasis.
Cassava is one of the world three yampi class, extensively cultivates in subtropical and tropical zones, and in China torrid areas, cassava is the fifth-largest crop being only second to paddy rice, sweet potato, sugarcane and corn, has strong adaptability, plantation region feature widely.Other cash crop, utilize cassava fermentative production cost low, can accomplish not strive grain with people relatively.Therefore develop with cassava is that fermenting raw materials is produced the novel process of beta-amylase and had very tempting commercial promise.
Wild-type B. subtilis involved in the present invention produces the outer heat-resisting beta-amylase of born of the same parents not only temperature, pH wide accommodation and heat-resisting acid resistance is good, illustrates tempting prospects for commercial application and commercial value.
Summary of the invention
Originate as plant for current beta-amylase main flow, but be difficult to carry out stable supply, and turnout is limited.On the other hand, microbe-derived beta-amylase, because productivity is low, thermotolerance is poor, production cost is high, seldom can realize practical.The invention provides a kind of bacterial strain and low-cost fermentation processing method thereof of producing thermotolerance beta-amylase, comprise the screening of bacterial strain, the optimization of substratum, the optimization of fermentation condition and zymology Quality Research, utilize cassava for the method for fermenting raw materials production beta-amylase.The invention provides wild-type B. subtilis and utilize the outer beta-amylase of cassava fermentative production born of the same parents, there is thermotolerance, cost is low, output is high, the advantage of good product performance, for suitability for industrialized production lays the foundation.
One of the object of the invention obtains a kind of bacterial strain Bacillussubtilis6-7 producing thermotolerance beta-amylase.This bacterial strain is (related application number: 201010167019.7) disclose, and have submitted preservation before this and to prove and survival proves in applicant's patent application before this.Obtain as drawn a conclusion to the correlative study of this bacterial strain:
1. substratum and culture condition
LB substratum: Tryptones 1%, yeast extract 0.5%, sodium-chlor 1%, pH7.0
Starch solids substratum: Tryptones 1%, yeast extract 0.5%, sodium-chlor 1%, Zulkovsky starch 1% and agar powder 2%, pH7.0
Primary dcreening operation substratum: Tryptones 1%, yeast extract 0.5%, sodium-chlor 1%, Zulkovsky starch 1%, pH7.0
Sieve substratum again: Tryptones 1%, yeast extract paste 0.5%, glucose 0.1%, Zulkovsky starch 0.5%, potassium primary phosphate 0.2%, bitter salt 0.05%, CALCIUM CHLORIDE DIHYDRATE 0.02%, pH7.0.
Two of the object of the invention is to provide a kind of subtilis B.subtilis6-7 to produce the processing method of heat-resisting beta-amylase, and makes with low cost, and raw material sources are extensive, comprise the optimization of substratum, the optimization of fermentation condition, is achieved through the following technical solutions, and step is as follows:
(1) basic medium: 2% glucose, 2% Tryptones, 0.5% Zulkovsky starch, 1% sodium-chlor, pH7.0, draws growth curve and produces enzyme curve (as accompanying drawing 1);
(2) C source is optimized, selects cassava to be main C source, and its optimal concentration is selected (as accompanying drawing 2);
(3) other compositions of basic medium are optimized: as Organic N source, inorganic N source, inorganic salt, buffer system etc.;
(4) Optimal Medium: Tapioca Starch 2%, bean cake powder 4%, 0.1% Secondary ammonium phosphate, 0.00139% ferrous sulfate, 0.6% Trisodium Citrate, 0.4% potassium primary phosphate, 0.0005% zinc sulfate, 0.0123% magnesium sulfate, 0.0111% calcium chloride, pH7.0;
(5) to fermentation culture conditions optimization: as culture temperature, initial pH, liquid amount, rotating speed, inoculum size, incubation time;
(6) shake flask culture conditions: this bacterium is in the 250ml shaking flask containing 50ml Optimal Medium, and initial pH is 7.0, inoculum size 4% (V/V), 160-190r/min, 37 DEG C of fermentation culture 60h.
(7) B.subtilis6-7 bacterial strain is cultivated in basic medium, shake-flask culture 60h, and enzyme is lived and reached 2200.0U/ml.Through C source, Organic N source, inorganic N source, inorganic salt, experiment of single factor and the orthogonal experiment Optimal Medium such as buffer system, optimum culture condition again, according to optimal culture conditions shake-flask culture 60h in Optimal Medium, the highest production of enzyme reaches 6724.0U/ml, improves 206.45% than before optimization.
Three of the object of the invention is the enzyme reaction conditions obtaining bacterial strain B.subtilis6-7 peak enzymolysis-ability starch, obtains following result:
(1) this enzyme thermal adaptation a wider range, optimum temperature is 65 DEG C, and the temperature preserved at 60 ~ 70 DEG C is better, and more than 70 DEG C to preserve long-time temperature stability poor, but to preserve in 30min remnant enzyme activity still higher (as accompanying drawing 3).
(2) this enzyme optimal reaction pH value is 6.0, all has high enzyme vigor (as accompanying drawing 4) between pH5-7.
(3) this enzyme molecular weight is at about 65KD.
(4) act on: the α-Isosorbide-5-Nitrae glycosidic link acting on polyose and oligosaccharide kind, maltose is dissociated;
(5) substrate specificity specificity: hydrolysis Zulkovsky starch, amylose starch, amylopectin, glycogen etc., can not be hydrolyzed beta-cyclodextrin, trisaccharide maltose.
Beneficial effect of the present invention: the present invention obtains the Bacillus subtilis 6-7 of the heat-resisting acidproof beta-amylase of high yield by screening from the bacterial strain in laboratory preservation and the soil sample of collection, after fermentation optimization, acquisition is with cheapness and the Tapioca Starch of wide material sources is the method that nitrogen source fermentation produces beta-amylase for carbon source and bean cake powder, greatly reduces production cost.
Accompanying drawing explanation
The growth of Figure 1B .subtilis6-7 in basic medium and produce enzyme curve
Fig. 2 different carbon source produces enzyme effect to B.subtilis6-7
The beta-amylase vigor of B.subtilis6-7 at Fig. 3 differential responses temperature
The beta-amylase vigor of B.subtilis6-7 under Fig. 4 differential responses pH
Embodiment
Below, the present invention will be further detailed by embodiment, but it is not limited to these embodiments any one.
Embodiment 1: the screening of producing amylase strain
The separation and purification of genus bacillus: 25 parts of soil samples of getting collection make soil supension through 80 DEG C of thermal treatment 15min, 37 DEG C of enrichment culture 24h, 24h is cultivated for 37 DEG C in gradient dilution coating LB solid plate, single bacterium colony is isolated in line again, until obtain purebred, and by purebred strain number and slant preservation, the preservation of glycerine frozen pipe.Then spore staining, gram stain microscopy, determine that the bacterium colony screened is genus bacillus.The bacterial classification of Laboratories Accession is reactivated, thermal treatment simultaneously, then coat on LB solid plate, filter out genus bacillus, slant preservation, glycerine frozen pipe preservation numbering
Produce amylase strain primary election: genus bacillus dibbling separation and purification obtained, in starch solids substratum, is cultivated 24h, 0.05% iodine staining bacterium colony for 37 DEG C, observed the diameter of transparent circle and bacterium colony circle, and record.Colony inoculation large for hydrolytic circle (D)/colony diameter (d) ratio is cultivated 12h in LB substratum, and glycerine frozen pipe is preserved in-40 DEG C.
Primary dcreening operation: hydrolytic circle and the large colony inoculation of colony diameter ratio are activated in LB substratum, transfers in primary dcreening operation substratum, in 37 DEG C, 160rpm cultivates 24h, measure fermentation broth enzyme and live.
Multiple sieve: higher bacterial strain of being lived by primary dcreening operation enzyme, through the activation of LB substratum, is transferred in multiple sieve substratum, in 37 DEG C, 160rpm cultivates 24h, measures fermentation broth enzyme alive.Determine that enzyme the highest Strain Designation alive is 6-7, as research starting strain.
Embodiment 2: the mensuration of amylase activity
The mensuration that amylase enzyme is lived adopts Bernfeld improved method, i.e. DNS measuring method, utilizes beta-amylase and Zulkovsky starch effect, the reductibility of release maltose residue, 3,5-dinitrosalicylic acid is reduced into 3-amino-5-NITROSALICYLIC ACID, then uses the content of spectrophotometric determination reducing sugar.1ml2% Zulkovsky starch solution is added in 25ml colorimetric cylinder, 1mlpH6.0 citrate buffer solution, in 40 DEG C of water-bath 5min, adds the crude enzyme liquid 1ml of dilution certain multiple, mixing, react in 40 DEG C of water-bath 30min, after reaction terminates, add 2ml3,5-edlefsen's reagent, 10ml is boiled in boiling water, flowing water cools, and deionized water is settled to 25ml, under 540nm, measure light absorption value.
The definition of Mei Huo unit: 70 DEG C, under the above-mentioned reaction conditions of pH6.0, it is 1U that 1ml liquid enzymes 30min hydrolyzed starch produces the enzyme amount needed for reducing sugar being equivalent to 1 μm of ol maltose, represents with U/ml.
The optimization of embodiment 3 fermention medium and culture condition
(1) drafting of growth curve and product enzyme curve: at basic medium (2% glucose, 2% Tryptones, 0.5% Zulkovsky starch, 1% sodium-chlor, pH7.0) growth curve of 6-7 bacterial strain in basic medium is measured, as shown in Figure 1.This bacterium, at 37 DEG C, is cultivated under the condition of 160r/min (rotary shaker) after 12 hours and enters logarithmic phase, and 40h enters and grows stationary phase, and beta-amylase adds up to reach the highest enzyme 2200.0U/mL alive at 60h.It can thus be appreciated that beta-amylase synthesis and thalli growth half coupled mode.
(2) optimization in C source: change the glucose in basic medium into Zulkovsky starch, ground rice, Semen Maydis powder, Tapioca Starch, molasses, chitosan, maltose, alpha-lactose, glucose, beta-cyclodextrin, plant 12h in age, inoculum size 4%, 37 DEG C, 160r/min fermentation culture.Cassava is that the enzyme activity in C source can reach 5120.5U/mL, 2.55 times of to be Zulkovsky starch be C source, 2.33 times of to be glucose be C source, 2.03 times of to be Semen Maydis powder be C source, 1.87 times of to be ground rice be C source, namely illustrates that cassava is that the fermentation of C source is with the obvious advantage
(2) medium optimization: change the N source of basic medium into other Organic N source, determine optimum N source and optimal concentration thereof; Add different inorganic salt, determine the inorganic salt promoting to produce enzyme; Add different inorganic nitrogen, determine the suitableeest inorganic nitrogen and add concentration; Add different buffer system, determine optimized buffer system; According to experiment of single factor, determine to affect large factor, design 4 factor 3 water-glasses, carry out orthogonal optimization, obtain optimal medium combination.
(3) optimization of fermentation culture conditions: the optimization carrying out culture condition in Optimal Medium, as culture temperature, rotating speed, initial pH, liquid amount, inoculum size etc., determine that optimal culture condition is: 37 DEG C, 190r/min, initial pH7.0,50mL liquid amount is in 250mL triangular flask, 4% inoculum size
(4) enzyme activity determination: this bacterium carries out shake flask fermentation in the 250ml shaking flask containing 50ml Optimal Medium, inoculum size 4%, 37 DEG C, 190r/min cultivates 60h, centrifuging and taking fermented supernatant fluid, record enzymic activity and reach 6724.0U/ml, improve 2.05 times than 2200.0U/mL before optimization.
Embodiment 4: heat-resisting beta-amylase zymologic property
(1) temperature is on the impact of enzymic activity
Same reaction system, measures this amylase enzyme activity respectively at 30 ~ 80 DEG C, result as shown in Figure 2: this enzyme all shows higher enzymic activity between 55 ~ 75 DEG C, and proper temperature a wider range of this enzyme is described, its optimum temperature is 65 DEG C.
(2) pH is on the impact of enzymic activity
Same reaction system, under 3-8.0, survey this enzyme enzyme respectively live, as shown in Figure 3, this enzyme enzymic activity when pH5-7 is higher, and the suitableeest action pH is 6.0 for result.
(3) mensuration of amylase protein molecular size range
The crude enzyme liquid obtained fermenting is by SDS-PAGE analyzing proteins molecular weight, and this enzyme molecular weight is at about 65KD.
Claims (2)
1. the beta-amylase of Bacillus subtilis 6-7 production, it is characterized in that, the optimal reaction pH of this enzyme is 6.0, is to use under the condition of 4.5-7.5 at pH; And there is following characteristic:
(1) this enzyme molecular weight is at about 66kDa;
(2) act on: the α-Isosorbide-5-Nitrae glycosidic link acting on polyose and oligosaccharide kind, generate free beta-maltose;
(3) substrate specificity specificity: hydrolysis Zulkovsky starch, amylose starch, amylopectin, glycogen, can not be hydrolyzed beta-cyclodextrin, trisaccharide maltose;
(4) be hydrolyzed the product of Zulkovsky starch, be mainly maltose, be secondly a small amount of trisaccharide maltose, glucose, there is no maltotetrose, maltopentaose, MALTOHAXAOASE;
(5) temperature of reaction is that 65 DEG C of enzyme activities are the highest, has thermotolerance;
Wherein Bacillus subtilis 6-7 is preserved in China typical culture collection center, and deposit number is: CCTCCM209200.
2. the fermentation method for producing of beta-amylase as claimed in claim 1,
(1) seed culture: seed culture medium is 1% peptone, 0.5% yeast extract paste, 0.5% Zulkovsky starch, 1% sodium-chlor, pH7.0, be in the Bacillussubtilis6-7 bacterial strain access seed culture medium of CCTCCM209200 by the deposit number of-40 DEG C of glycerine frozen pipes preservation, cultivate 12h at 37 DEG C and 160r/min shake flask, then be forwarded in fresh seed culture medium and cultivate 12h as secondary seed solution;
(2) fermention medium: Tapioca Starch 2%, bean cake powder 4%, Secondary ammonium phosphate 0.1%, ferrous sulfate 0.00139%, Trisodium Citrate 0.6%, potassium primary phosphate 0.4%, zinc sulfate 0.0005%, magnesium sulfate 0.0123%, calcium chloride 0.0111%, pH7.0; Described Tapioca Starch is that cassava block is directly broken into pieces, and mechanical grinding becomes powder, and 40 orders sieve, and directly takes Tapioca Starch and put into triangular flask, without enzymolysis or acidolysis process during substratum preparation;
(3) sterilizing: 121 DEG C of sterilizing 25min;
(4) conditions of flask fermentation: the fermention medium of the bottled 50ml of 250ml triangle, inoculum size is 4% volume ratio, 37 DEG C, 190r/min shaking table shaking culture, fermentation 64h.
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| CN101851598A (en) * | 2010-05-10 | 2010-10-06 | 江南大学 | Environmentally-friendly breeding of bacillus subtilis for producing 2,3-butanediol by fermentation with glucose substrate |
| CN102127529A (en) * | 2010-11-29 | 2011-07-20 | 南宁邦尔克生物技术有限责任公司 | Method for recombinant expression of beta-amylase in bacillus subtillis in integrated mode |
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| CN101851598A (en) * | 2010-05-10 | 2010-10-06 | 江南大学 | Environmentally-friendly breeding of bacillus subtilis for producing 2,3-butanediol by fermentation with glucose substrate |
| CN102127529A (en) * | 2010-11-29 | 2011-07-20 | 南宁邦尔克生物技术有限责任公司 | Method for recombinant expression of beta-amylase in bacillus subtillis in integrated mode |
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